Isolation, identification and characterization of endophytic fungi of Bambusa oldhamii munro applied as antagonists to Pyricularia oryzae 1

Endophytic fungi, in addition to improving plant development, may produce antimicrobial substances that can inhibit pathogens. The objective of this study was to isolate and characterize endophytic fungi of Bambusa oldhamii and evaluate in vitro antagonism to Pyricularia oryzae . Fungal isolates of B. oldhamii shoots were purified and identified by nuclear ribosomal DNA (nrDNA) including internal transcribed spacer regions (ITS1 and ITS4). These isolates were then confirmed by phylogenetic analysis of the ITS1 and ITS2 sequences and morphological analysis. The isolates were evaluated in in vitro experiments of direct and indirect antagonism (volatile production). Five fungal isolates were identified and named as follows: isolate 27 ( Arthrinium sp.); isolate 29 ( Acrocalymma sp.); isolate 122 ( Botryobambusa fusicoccum ); isolate 711 ( Phoma sp.) and isolate 712 ( Phoma sp. ) . We found that the area of the P. oryzae colony was reduced by more than 80% by all endophytic isolates in this study, with emphasis on isolate 122, with a 96% reduction in area of the P. oryzae . These results were considered promising and will serve as a foundation for future studies on induction of resistance to P. oryzae .


INTRODUCTION
Endophytic fungi are microorganisms that live within plants for at least a part of their life cycle without causing any visible manifestation of disease. This relationship benefits the host in several ways, protecting it from biotic and abiotic stress (Selim et al. 2012). This relationship includes secretions of substances by endophyte, which are biologically active metabolites which promote antagonistic action against phytopathogens (Zhang et al. 2006;Sudha et al. 2016).
Bambusicolous fungi (formerly fungorum bambusicolorum) are species that have association with bamboo species (Dai et al. 2017). This group is estimated to include more than 1,450 species in approximately 115 genera around the world. They include ascomycetes, basiomycetes, coleomycetes, hyphomycetes and anamorphic fungi (Shukla et al. 2016;Dai et al. 2017). These fungi, are important because of the compounds they produce, such as metabolites with antioxidant, antimicrobial and antitumor properties (Oyetayo et al. 2009). Therefore, research involving the identification and characterization of species associated with bamboo is necessary in order to use these compounds in the biocontrol of pathogens in cultivated plants in the future (Oyetayo et al. 2009;Ebada et al. 2011;. Currently, there is high demand for biological agents for disease control in various crops. Excessive use of pesticides for pathogen control in agriculture generates a series of environmental problems such as soil and groundwater contamination by heavy metals (Lu et al. 2015). Rice blast is the major disease in rice cultivation, caused by the fungus Pyricularia oryzae B. C. Couch [anamorph -Magnaporthe oryzae Cavara]. Under Isolation, identification and characterization of endophytic fungi of Bambusa oldhamii munro ... Rev. Ceres, Viçosa, v. 67, n.4, p. 296-305, jul/aug, 2020 favorable conditions, rice blast can cause crop losses of up to 100% (Prabhu et al. 1992;Prabhu & Filippi 2001;Prabhu et al. 2009). For disease control, in addition to the use of fungicides, integrated management by means of farming practices is also necessary (Prabhu & Filippi 2001).
P. oryzae has a cell wall composed of melanin, which is important for the mechanism of pathogen infection, which involves the formation and peg penetration of the appressorium. Melanin plays a structural part in strengthening the appressorium and forms an impermeable layer to prevent leakage of osmolytes, which generate the substantial internal turgor (Wilson & Talbot 2009). Thus, the loss or blockade of melanin in the colony also implies the loss of pathogenicity (Yan & Talbot 2016;Oses-Ruiz et al. 2017). Thus, the development of biological control techniques that provide blockade of melanin in P. oryzae colonies could provide improvements in rice production. Research involving antagonist relations between endophytes and pathogens is necessary for future search for novel natural products (Nisa et al. 2015), as well as the identification of bioactive compounds that could be used in research involving induction of resistance in plants (Busby et al. 2016).
When it comes to interactions involving plants and microorganisms, fungal endophytes are involved in multiple balanced antagonisms. In planta, endophytic fungi must deal with multiple organismal interactions. This relationship involves anti-fungal metabolites produced by endophytic fungi, which have the function of maintaining balances of antagonisms with microbial competitors, resulting in a compatible multipartite symbiosis (Schulz et al. 2015). Therefore, research involving identification of endophytic fungi and their antagonisms between P. oryzae are necessary in order to develop natural products in the future that may be inhibitors of pathogen growth under natural conditions, or even use these endophytes as growth resistance inducers. Thus, the objective of this study was to identify and morphologically characterize endophytic fungi of Bambusa oldhamii Munro and to evaluate possible antagonism to P. oryzae, for use in future studies on resistance and plant growth promotion.

Isolation of endophytic fungi
Secondary shoots of B. oldhamii measuring 55 -60 mm were collected in March 2016 at the Bamboo Collection of the Federal University of Goiás (Universidade Federal de Goiás), located in Goiânia, Goiás state, Brazil. The shoots were sectioned into 50 mm fragments, surface washed with neutral detergent solution (50 µL per 100 mL of water) and, after triple washing, immersed in sodium hypochlorite solution (0.0025% active chlorine) and stored at 5 ºC for 24 h. In a laminar flow chamber, asepsis was performed with Tween 80 ® solution (50 µL per 100 mL) for 10 min, 70% ethanol for 5 min and sodium hypochlorite (2.5% active chlorine) for 15 min, followed by triple washing in deionized and autoclaved water after each step. After asepsis, the shoots were immersed in a 5% solution of Plant Preservative Mixture ® (PPM) biocide for 24 h. Then, the samples were cut into 30 mm fragments and transferred to test tubes containing MS medium (Murashige & Skoog, 1962) supplemented with 30% sucrose, 0.45 µM thidiazuron (TDZ) (Lin et al. 2007), 0.2% PPM ® and 103.5 µM kanamycin. The test tubes containing the shoots and culture medium remained in the dark for 15 days and then were incubated for 15 days in the growth room under cold white light with light intensity of 35 µmol m -2 s -1 and a photoperiod of 16 h light.
After 30 days, the fungal colonies grown in the ten test tubes were transferred twice consecutively to Petri dishes containing potato dextrose agar (PDA) medium for purification of the colonies. For this, the mycelium suspension was used. This suspension was spread on Petri dishes (containing 30 ml of PDA medium) with the aid of a glass stick. The color, texture and growth form of the mycelium were observed for purification, as well as the morphological characteristics in microscopic, in order to confirm the purification. In all, five distinct colonies were observed, and the dishes were stored in a growth room at 28 ± 1 ºC with a photoperiod of 12 h of light.

DNA extraction and isolate identification
The mycelium was scraped and the DNA was extracted from the fungal isolates (Dellaporta et al. 1983). The extraction quality was measured using a NanoDrop TM 2000 spectrophotometer and, after diluting the DNA to 20 ng/ µl, the samples were amplified and sequenced by BPI Biotecnologia Pesquisa e Inovação. The DNA samples were amplified using primers for sequences from internal transcribed spacer genomic regions (ITS1 and ITS4) (White et al. 1990;Manter & Vivanco 2007). The amplified samples were evaluated on 2% agarose gels, stained with 0.03% (v/v) UniSafe Dye, visualized under UV light and quantified by fluorometry (Qubit, ThermoFisher). For the sequencing reaction, a BigDye® Terminator v3.1 and Cycle Sequencing Kit (Applied Biosystems) were used. For the precipitation reaction, ethanol/EDTA/sodium acetate were used, according to the protocol suggested by the manufacturer. Automated capillary electrophoresis sequencing was performed on a ABI3500 Genetic Analyzer (Applied Biosystems).
For molecular identification of the isolates, the resulting sequences were subjected to a BLAST search with the NCBI database (http://www.ncbi.nlm.nih.gov/) and were confirmed by phylogenetic analysis of the ITS1- Rev. Ceres, Viçosa, v. 67, n.4, p. 296-305, jul/aug, 2020 5.8S-ITS2 regions. These methods were combined with morphological analysis for isolates in which conidiogenesis was observed in the culture media (described in the next section) based on dichotomous keys and updated revisions (Barnett & Hunter 1998;Shukla et al. 2016;Dai et al. 2017). The ITS 1 and 2 sequences were aligned via ClustalW using the software MEGA version 7 (Kumar et al. 2016). The tree was constructed using the Maximum Likelihood method (Nei & Kumar 2000). Bootstrap analysis (10,000 replicates) was used as statistical support. The initial trees were obtained by applying the Neighbor-Join and BioNJ algorithms (Saitou & Nei 1987). Only sequences published in journals were used, and the sequences resulting from this study were deposited into GenBank.
The growth rate of the isolates (radial growth) was calculated as the mean value obtained with the equation: PRG -CRG/CRG, where PRG refers to the radial growth of the previous evaluation and CRG to the current radial growth. The evaluation was performed every 2 days, and the quantitative data were subjected to analysis of variance and the means were compared by the Tukey test at the 5% significance level.
The evaluation of colonies, color and the presence or absence of conidiogenesis was performed. In addition, the mycelium of isolates was observed under an optical microscope to characterize the hyphae and conidia. For the microscopic observation of the isolates, slides were prepared with fungal mycelium, 10 µL of deionized and distilled water and 1 µL of methylene blue at a concentration of 0.1% to stain hyaline hyphae. Observation was performed using an optical microscope coupled with a digital camera.

Antagonism between endophytic isolates of bamboo and the fungus Pyricularia oryzae
Mycelial discs of bamboo fungal isolates measuring 7 mm in diameter were placed in dishes containing 30 mL of PDA culture medium according to the circle method (Da Luz, 1990), in which discs of the two isolates are positioned equidistant from the dish edges. The dishes containing only P. oryzae mycelial discs placed in the center of the dish were used as controls. The dishes were incubated (28 ± 1 ºC with a photoperiod of 12 h light) until full growth of the control was achieved (10 days). Next, the radial mycelium growth of P. oryzae on each dish was calculated (Cattelan 1999).
Mycelial discs measuring 5 mm in diameter of each endophytic isolate were placed in the center of Petri dishes (90 x 90 mm) containing 30 mL of PDA culture medium. P. oryzae mycelial discs grown in PDA culture medium were also placed in the same way in another Petri dish. The lids of all Petri dishes were removed, and the bases of all dishes containing endophytic isolates (antagonist) and the pathogen (P. oryzae) were combined, forming an experimental unit. Dishes containing only P. oryzae mycelial discs were used as controls. The dishes were incubated (28 ± 1 ºC with a photoperiod of 12 h light) until full growth of the control was achieved. Next, the radial mycelium growth of P. oryzae on each dish was calculated (Dennis & Webster 1971;Cattelan 1999).
In both the direct and indirect antagonism experiments, the experimental design was completely randomized, with 7 treatments composed of 7 fungal isolates and 4 replicates. The data were subjected to Box-Cox transformation to meet the assumptions of the analysis of variance, and the means were compared by the Tukey test at the 5% significance level. The data were analyzed using R version 3.4.3 (R Core Team 2017).
The percentage of growth inhibition (PGI) in direct and indirect antagonism was calculated according to the equation (ND -ED)/ND x 100 (Shen et al. 2012), where ND refers to the diameter of the negative control, i.e., the P. oryzae isolate grown without the interference of any antagonist, and ED refers to the diameter of P. oryzae when subjected to the antagonism of a B. oldhamii endophytic isolate.
Conidiogenesis of isolate 711 was observed in CMA medium. The conidia were of the pycnidia type (Figure 2), measuring 31.87 -100 µm x 81.25 -100 µm (width x length), and had glabrous, globose, blackish brown ostioles (dimensions 12.5 -87.26 µm) and a brown peridium. Hyphae were septate, uni-or multinucleate, with dimensions of 9.3 -19.7 µm. The CMA culture medium also favored the formation of chlamydospores in isolate 712 (Phoma sp.). These measured 28.13 x 25.73 µm, had a red-brown catenulate peridium, and were glabrous, globose or subglobose, individual or clustered (Figure 2). The hyphae were septate, glabrous, uni-or multinucleate, measuring between 6.5 -35.5 µm.  Among the different culture media evaluated, the CMA medium significantly favored the growth of isolates 711 and 712. However, no significant difference was observed between this culture medium and the PDA and TSA media regarding colonies of isolate 122. For isolates 27 and 29, all culture media favored colony growth, with no significant difference among them.
Isolate 122 presented faster growth than the other fungi (Table 2) and achieved full growth in the Petri dish (90 x 90 mm) after approximately 48 h in PDA medium. By contrast, isolate 29 (Acrocalymma sp.) presented the slowest mycelial growth in the culture evaluated media (Table 2).

Molecular identification
After purification of the mycelium, five isolates were obtained, which were called isolates 27, 29, 122, 711 and 712. Amplification of the ITS1-5.8S-ITS2 region with primers ITS1 and ITS4 produced fragments of approximately 580 bp for isolates 27 and 122; 510 bp for isolate 29; 500 bp for isolate 711; and 550 bp for isolate 712.
Analysis of the ITS1-5.8S-ITS2 homologous sequences of each endophytic isolate was conducted, resulting in a phylogenetic tree with four families. The bootstrap percentage of the taxa is shown next to the branches ( Figure  3). A separate branch was observed for isolate 29 (Acrocalymma sp.) within its respective taxon, indicating a previously unidentified species of Acrocalymma ( Figure  3). The ITS1 and ITS2 sequence data of isolates were submitted in GenBank. The accession numbers for the nucleotide sequences were: MN381104 for isolate 27; MN381026 for isolate 29; MN381103 for isolate 122; MN381110 for isolate 711; and MN383185 for isolate 712.

Antagonism between endophytic fungi and Pyricularia oryzae
The mycelial growth of P. oryzae was reduced by all isolates in direct antagonism, with emphasis on isolate 122 (B. fusicoccum) (Figure 4), showing a probable competition-type interaction. Similarly to direct antagonism, the volatile components ( Figure 5) produced by the isolates under study reduced the area of the P. oryzae colony, with reductions of over 80% (Table 3 and Figure 6), showing a probable inhibition-type interaction. The phenotypes of P. oryzae colonies were altered, with loss (total or partial) of the black color (melanin).

DISCUSSION
Identification of the genera of endophytes of B. oldhamii was successful. Isolate 27 (Arthrinium sp.) belongs to a genus that includes saprophytic species with sexual forms called Apiospora. This species is reported to be a pathogen of bamboo (Martínez-Cano et al. 1992), an endophyte (Ramos et al. 2010) and a lichen (He & Zhang 2012). Isolate 29 was identified as belonging to the genus Acrocalymma, which includes saprophytic and phytopathogenic species, and includes A. medicaginis (Alcorn & Irwin 1987). Isolate 122 was identified as belonging to the genus Botryobambusa, whose sole member is the saprophyte B. fusicoccum. This species is commonly associated with bamboo, especially of the genus Bambusa (Liu et al. 2012). Isolates 711 and 712 were identified to the genus Phoma, a diverse genus that includes, in addition to growthpromoting microorganisms (Hamayun et al. 2009), phytopathogens, entomopathogens, soil saprophytes, aquatic saprophytes and opportunistic human pathogens (Rai et al. 2009). Endophytic fungi of B. oldhamii were previously reported by Pasqualini et al. (2019), which report that research associated with the discovery of these isolates may increase the current knowledge about the diversity of fungi associated with bamboo. They report the isolation of phylogenetically similar isolates to those found in the present paper, which include: Phoma herbarum, Arthrinium kogelbergense, Arthrinium arundinis, Fusarium graminearum, Wojnowiciella leptocarpi, and Fusarium sp. The phylogenetic position of isolates 711 and 712 (Phoma sp.) can still be investigated in the future. In fact, the taxonomic circumscription of the Phoma genus has been complex (Chen et al. 2015;Valenzuela-Lopez et al. 2018). Phoma is ambiguously defined and is subdivided into nine sections based on morphological characters. These sections are classified into four different families, with strains genetically similar to P. herbarum belonging to the family Didymellaceae, which includes the genus Epicoccum (Jayasiri 2017). For this reason, Phoma constitutes a polyphyletic group (Gruyter et al. 2009). Despite the efforts of researchers to characterize the group (Chen et al. 2015), there are still gaps in its systematics, both with regard to morphological and molecular characters because many species still remain in nonspecific clades (Aveskamp et al. 2008). Thus, sequencing of additional genomic regions and of isolates similar to isolates 711 and 712 are necessary to better understand the phylogenetic relationships and species boundaries.
The mycelial growth rate and conidiogenesis of the endophytic isolates were influenced by the different culture media evaluated. The nutrient composition of a given culture medium is an important factor that influences the growth rate, as it allows the fungi to grow without restrictions and express phenotypes (Meletiadis et al. 2001). Sharma and Pandey (2010) used different fungal isolates in different culture media and found that the best culture medium for conidiogenesis of the genera Chaetomium, Fusarium, and Penicillium was LCA medium (glucose, 1 g; KH 2 PO 4 , 1 g; MgSO 4 .7H 2 O, 0.2 g; KCl, 0.2 g; NaNO 3 , 2 g; yeast extract, 0.2 g; agar, 13 g; distilled H 2 O, 1 L) which is a culture medium with reduced amounts of nitrogen and carbohydrates, which may induce conidiogenesis (Miura & Kudo 1970). In the present study the CMA medium was evaluated, which contains significant amounts of carbohydrates, vitamins and salts. However, not sufficiently rich in amino acids, which are sources of nitrogen (Gernah et al. 2011). Thus, probably the conidiogenesis was influenced by the lack of nitrogen in the culture medium. However, further studies should be performed to substantiate this claim.
Using the direct antagonism assay, we found that the area of the P. oryzae colony was reduced by more than 80% by all endophytic isolates in this study. The interactions involved with biological control include competition, which comprises direct antagonism and is the result of physical contact between the biocontrol organism and the pathogen, in which there is a high degree of selectivity for the pathogen via the mechanism or mechanisms expressed by the biocontrol microorganisms (Harman et al. 2004). These mechanisms include the secretion of enzymes with antimicrobial activity, including PPOs, chitinases, and glucanases (Brzezinska & Jankiewicz 2012;Naznin et al. 2014). Probably, this metabolite secretion inhibited the production of melanin by P. oryzae colonies, when they were submitted to treatments with endophytic isolates. Under natural conditions, P. oryzae needs melanin for activation of pathogenicity mechanisms (Carvalho et al. 2015;Oses-Ruiz et al. 2017). Therefore, further in vivo research should be performed to confirm the antagonistic potential of the isolates of the present study.
Using the indirect antagonism assay, we also observed high inhibition of the P. oryzae colony area by all isolates studied (above 80%). This result indicates that these endophytes are likely stimulators of defense mechanisms in plants, which can be clarified in future studies on resistance induction (Harman et al. 2004;Silva et al. 2004).
The antagonistic activity of endophytes of bamboo was reported by (Shen et al. 2012), who investigated the antibiosis of endophytes isolated from a Phyllostachys species (Cladosporium sp., Curvularia sp., Penicillium sp., and Didymella sp.) to Pleospora herbarum and Botryotinia fuckeliana, the latter considered a bamboo pathogen (Mohanan 1997). In addition to these, the authors also reported antibiosis of these endophytes with Candida albicans and Staphylococcus aureus, both clinical pathogens.  Rev. Ceres, Viçosa, v. 67, n.4, p. 296-305, jul/aug, 2020 No inhibition halo was observed in the direct antagonism assay between P. oryzae and isolate 27 (Arthrinium sp.) or isolates 29 (Acrocalymma sp.) and 122 (B. fusicoccum) (Figure 4). However, species of the genus Arthrinium have been reported to have antibacterial (Calvo et al. 1999;Miao et al. 2006), antifungal (Bloor 2008;Ramos et al. 2010) and antiviral  activity. For this reason, it is suggested that isolate 27 characterized in this study may be the object of future research with other plant species as well as in the interaction with other bacterial pathogens.
The isolate 122 (B. fusicoccum) presented basic characteristics as an antagonist against P. oryzae. The other isolates were also identified as promising for antagonism, including isolate 711 (Phoma sp.) and 712 (Phoma sp.). However, further in vivo studies are required.